Oxygen and nutrients required for the development of living tissues are carried to the tissues through the blood vessels whereas waste substances are carried away from the tissues via the blood vessels as well. While angiogenesis, growth of new blood vessels from pre-existing vessels, is a physiological phenomenon in embryogenesis, wound healing, and during menstruation in the female reproductive system, it emerges pathologically in inflammatory diseases such as arthritis, chronic inflammation, inflammatory bowel diseases, and psoriasis and such cancers of various tissues as breast, bladder, colon, lung, neuroblastoma, melanoma, kidney, pancreas, uterus, cervix, and glioblastoma as well as in ophthalmologic diseases such as age related macular degeneration.
The significance of angiogenesis in in vivo formation, development, and metastasis of solid tumors was first asserted in 1971 (Folkman J., 1971, N Eng J Med., 285, 1182-1186). Angiogenesis emerges through the proliferation of capillary endothelial cells (Risau W, 1997, Nature, 386, 671-674). As with all biological events, the organism responds to angiogenic stimulation by secreting antiangiogenic factors. Proangiogenic factors and antiangiogenic factors are at equilibrium under normal conditions and when this equilibrium is disturbed against antiangiogenic factors, angiogenesis starts. Vascular Endothelial Growth Factor (VEGF) was defined by Ferrara et al. in 1989 (Ferrara and Henzel, 1989 Biochem Biophys Res Commun 161, 851-858). VEGF plays a key role in angiogenesis (Ferrara N et al., 1996, Nature, 380: 439-442). Experiments on mice showed that the only lack of allele concerning VEGF resulted in early embryonic lethality due to serious vascular problems (Carmilet P. et al., 1996, Nature, 380, 435-439). VEGF is a heparin-binding homodimeric basic protein bound with disulfide bond of 45 kDa in weight. Such various sub-groups of it as VEGF-A, VEGF-B, VEGF-C, VEGF-D ve VEGF-E have been defined so far. In mammals, VEGF-A has isoforms such as VEGF121, VEGF165, VEGF189, VEGF206 and VEGF145 based on the number of amino acids. Among these isoforms, VEGF165 is the predominant one (Ferrara N., et al., 2009, Arterioscler. Thromb. Vasc. Biol., 29, 789-791,).
VEGF shows its intracellular effects by binding to tyrosine kinase receptors in cell membranes. VEGF Receptors contain two portions. The first section is the intracellular portion containing a tyrosine-kinase domain. The second portion is the extracellular region comprising 5 to 7 immunoglobulin-like structures that contain ligand binding regions (Ferrara N. et al., 2003, Nat. Med. 9:669-676). In addition to identification of Flt-1 (VEGFR-1), flk-1/KDR (VEGFR-2), and Flt-4 (VEGFR-3) as VEGF receptors, receptor structures termed neuropilin-1 and neuropilin-2 expressed at the endothelial cell surface and that has a low binding characteristic to VEGF-A have been defined recently. KDR (kinase-insert-domain-containing receptor) was defined by Terman et al. by patent numbered PCT/US92/01300 in 1991 (Terman et al., 1991, Oncogene 6:1677-1683). Flk-1 sequence was shed light upon by sequencing method in 1991 (Mathhews W et al., 1991, Proc. Natl. Acad. Sci. U.S.A, 88:9026-9030). These studies have demonstrated that KDR is the human analog of FLK-1 receptor. In addition, KDR and FLK-1 receptors are also known as VEGFR2.
The most important of VEGF receptors is KDR (VEGFR-2), which is responsible for endothelial cell proliferation and chemotaxis (Ferrara N et al., 2003, Nat Med., 9, 669-676). VEGFR-2 (Kinase insert domain receptor/KDR) is expressed at high levels in vascular endothelial cells and hematopoietic cells (Asahara T., et al., 1997, Science, 275, 964-967; Ziegler B l. et al., 1999, Science, 285, 1553-1558; Peichev M., et al., 2000, Blood, 95, 952-958). The 7 immunoglobulin like domains present in the extracellular section of KDR enable the signals from the environment to be transducted to the cytoplasm of the cell. The intracellular section of KDR mediates intracytoplasmic signal transduction. Therefor, any molecule aiming to inhibit the interacion VEGF with KDR should target the 1-7 immunoglobulin-like extracellular domains of the receptor (LU D. ve ark., 2000, JBC, 275, 14321-14330).
Several studies conducted so far have found that VEGF increases in many types of cancers such as glioblastoma, colorectal cancer, non-small cell lung, pancreas, ovary, acute myeloid leukemia, multiple myeloma, Hodgkin's disease and non-Hodgkin's, and myeloma (Ranieri G et al., 2006, Curr Med Chem., 13, 1845-1857). Therefore, VEGF and VEGF receptors are among the priority targets in suppression of angiogenesis. It is possible to prevent function of VEGF by inhibiting from different angles the signal path triggered by binding of VEGF to transmembrane tyrosine kinase receptors on endothelial cells with structures developed against VEGF and VEGF receptors.
Although the binding affinity of VEGF to VEGFR-1 (Flt-1) is 50 times more as compared with VEGFR-2 (KDR), VEGF's angiogenic features, endothelial cell proliferation and its effect on chemotaxis, take place due to its relation with KDR (Cross M. et al., 2003, Trends in Biochemical Sciences; 28.488-494). It has been demonstrated that when a plasmid encoding VEGFR2 is given to pig aortic endothelial cells lacking VEGFR2, these cells go through mitosis and participate in chemotaxis (Shibuya M et al., 1990, Oncogene, 8:519-524). Some studies on mice showed that these animals lacked organized blood vessels in deficient or defective expression of VEGFR2. Shalaby et al. showed that mouse embryos lacking VEGFR-2 expression die during the early embryonic period due to the deficiency in the development of endothelial and hematopoietic progenitor cells (Shalaby F et al., 1995, Nature. 376 (6535):62-6). With this experiment, it has been shown that blocking the relation between VEGF and KDR is important in terms of suppression of angiogenesis.
In 1971, J. Folkman asserted that growth of a tumor is dependent on oxygen and energy resources carried by new capillaries that develop from the blood vessels located near the tumor and claimed that antiangiogenic attempts may be an effective approach in terms of preventing cancer development. The close association of tumor growth with angiogenic activity has led to investigation of angiogenic agents as additional options of treatment in cancer treatment. Demonstration of the fact that antibodies developed against VEGF suppress tumor growth in vivo (Kim K J et al., 1993, Nature 362: 841-844) has shown that VEGF antagonists may be used in treatment as inhibitors of tumor vascularization. Today, VEGF and VEGF receptors are among the priority objectives in the suppression of angiogenesis and thereby, in oncology (Ferrara N and Kerbel R. S., 2005, Nature. 438: 967-974)
Several anti-angiogenic strategies based on blocking VEGF/receptor relationship have been developed in recent years. Within this framework, such various structures as anti-VEGF antibodies that prevent VEGF/KDR interaction and/or suppress KDR signal transmission for the inhibition of angiogenesis and tumor (Kanai et al., 1998, J. Cancer 77, 933-936; Margolin et al., 2001, J. Clin. Oncol. 19, 851-856); anti-KDR antibodies (Zhu et al., 1998, Cancer Res. 58, 3209-3214; Zhu et al., 2003, Leukemia 17, 604-61 1; Prewett et al., 1999, Cancer Res. 59, 5209-5218); anti-VEGF immunotoxins (Olson et al. 1997, Int. J. Cancer 73, 865-870); ribozymes (Pavco et al., 2000, Clin. Cancer Res. 6, 2094-2103); soluble receptors (Holash et al., 2002, Proc. Natl. Acad. Sci. USA 99, 11393-11398); tyrosine kinase inhibitors (Fong et al., 1999, Cancer Res 59, 99-106; Wood et al., 2000, Cancer Res 60, 2178-2189; Grosios et al., 2004, Inflamm Res. 53(4):133-42); anti-VEGF-antisense (Forster et al. 2004, Cancer Lett. 20; 212(1):95-103); and RNA interference (Takei et al. 2004, Cancer Res. 64 (10):3365-70; Reich et al., 2003, Mol Vis 9:210-6).
Studies focusing on suppression of angiogenesis by targeting VEGF and receptors have intensified. Many strategies have been developed to this end. The rhuMab VEGF (Bevacizumab), recombinant human monoclonal VEGF antibody with antiangiogenic and anti-tumor activity (Monk B. J. et al., 2005, Gynecologic Oncology, 96, 902-905) and the monoclonal human antibody developed in 2006 by Wu Y et al. (Wu Y, et al., 2006 Clin. Cancer Res., 12(21), 6573-84) against VEGFR-1′e are the most important ones. Results indicating that VEGF-Trap glioma-animal models with VEGFR1 and VEGFR2 hybrid structures combined to human IgG1 constant region can be used in treatment of tumors in the beginning and advanced stages (Gomez-Manzano C. et al., 2008, Neuro-Oncology, 10, 940). Ranibizumab, 48 kDa, comprised of the Fab (antigen-binding) section of Anti-VEGF monoclonal antibody, renders lighter and thinner than the monoclonal antibody Bevacuzumab (148 kDa) it is derived from, and thereby, is able to pass through the internal membrane in intravitreal application and inhibits all VEGF isoforms (Gaudreault J. et al., 1999, Am Assoc Pharm Sci Pharm Sci Suppl, 1, 2142). Today, promising results have been obtained in the use of Ranibizumab for treatment in age related macular degeneration (Rosenfeld P. J., et al., 2006, N Engl J Med., 355, 1419-1431).
In addition to monoclonal antibody structures, the use of small molecules as VEGF antagonists through acting as VEGF receptor tyrosine kinase inhibitors is possible as well. In a study conducted by Bainbridge J. W. B. et al. in 2003 with peptide structures created by setting off from the exon 6 region, where interaction of the VEGF molecule with KDR take place, 7 amino acid structures that inhibit angiogenesis by blocking interaction of VEGF with its receptor were identified in vitro. Recently, sunitinib and sorafenib, two small molecule VEGF receptor tyrosine kinase inhibitors, have been started to be used in cancer treatments (KO J. S et al., 2009, Clin. Cancer Res., 15(6), 2148-2157; Jilaveanu L. et al., 2009, Clinical Cancer Research, 15, 1076).
In line with the developments in genetic engineering in recent years, formation of functional recombinant antibody fragments that mimic antigen recognition of the antibody molecule has been possible. Expressed as an antibody fragment, single chain variable fragment is constituted by binding of heavy chain variable region (VH) and light chain variable region (VL) via a peptide bridge. These antibody fragments are called single chain variable fragments (scFv) (U.S. Pat. No. 4,946,778 Lander et al.; WO88/09344, Huston et al.). As ScFv structures contain antigen-binding variable regions (Fv), they have the characteristic to provide the binding feature of the antibody molecule at a minimal structure. On the other hand, single domain antibody structures may be effective in binding to the target antigen structure.
In 1993, Jeffrey et al. showed that heavy chain was fundamental in digoxin binding of the antibody developed against digoxin (Jeffrey, P. D. et al. Proc. Nat. Acad. Sci., USA 1993, 90:10310-103149). It has been shown in a recent study that the “nanobody” structures consisting of heavy chain variable fragments of the antibody developed against the epidermal growth factor receptor (EGFR) prevents binding of Epidermal growth factor (EGF) to EGFR (Roovers R. et al., 2007, Cancer Immunology 15:303-317). ScFv's are presented on the surfaces of filamentous phages in phage display technology (WO 92/01047 Mc Cafferty et al.). It is possible to develop nanoantibody structures against VEGFR with this approach. However, rapid removal from circulation of nano antibody structures of approximately 15 kD leads to reservations in treatment applications. However, these nano antibody structures have been enabled to stay in the circulation for longer periods by restructuring them as 50 kD multivalent to ensure that they are provided with the feature to bind to albumin (Tijink B et al. 2008, Mol Cancer Therapy. 7(8):2288-2297).
The use of filamentous phages in phage display technology has brought about various advantages. Easy purification of phage particles from culture supernatant, accessibility to genetic and sequence data, and the opportunity to select the small number of phage clones that can identify the target antigen from among many antigens using the “biopanning” method are some of these advantages. Phagemid vectors that contain both bacteriophage replication origin and plasmid replication origin are preferred in order to take advantage of this convenience in phage display technology. The multiple cloning site (MCS) in these phagemid vectors is located at the start point of the gene that belongs to the sheath structural protein of the phagemid (e.g., gIII) Thus, the product obtained as a result of fusions made on sheath proteins can mimic the antibody located on the surface of B-lymphocytes existing in the immune system in the normal environment. However, this process is dependent upon how the amber stop codon located between the antibody gene and gIII will be read by the host bacteria. If phage supE grows on a suppressive host (e.g., E. coli TG1), the antibody fragment makes fusion to the minor sheath protein and stays on the phage surface. This structure on phage surface can act as a receptor that detects such foreign structures as B surface antibodies. If phage is grown on non-suppressive (sup−) E. coli strains (such as HB2151), then amber codon will be read as stop codon and the antibody particle will be secreted from the bacteria in soluble form. As such, mimicking of the antibodies synthesized from plasma cells with phagemid vectors become possible. The region comprised of 6 histidines on the phagemid vector, following the expression of cloned gene fragment to the multiple cloning site as dissolved in non-suppressor bacteria, enables easy purification of this recombinant protein from the environment using Zn++, Ni++ or Co++ charged affinity column (Weiner L. M, 1996, J. Mol. Biol., 255(1), 28-43).
Due to the technical convenience of the phage display technology, the scFv structure reflects a wide range of utilization opportunities in many disciplines. In contrast to the approximately 150 kDa size of the whole antibody molecule, the scFv structures of the antibody, which are approximately 30 kDa in size and lack the constant region and Fc parts of the antibody, are being used in today's medical researches focusing on diagnosis and treatment in increasing numbers (Bradbury A. R. M. et al., 2004, Journal of Immunological Methods, 290, 29-49).
The KDR 1.3 and 2.6 scFv antibody structures mentioned in the invention present antiangiogenic characteristics by blocking the intracellular signaling activity of VEGFR-2 by binding to the extracellular part of the VEGFR2 (1-7 immunoglobulin domain) on the cell surface and by inhibiting VEGF dependent cell proliferation.